Titanium dioxide nanoparticles (TiO2 NPs) are widely used as pigments in paints, food, and cosmetics. Annual production is estimated at 1.2 M metric tons. While TiO2 NP exposure through the skin or gut is considered harmless, inhalation into the lung by workers processing these materials remains a concern. Recent results from the Payne Lab, which relocated from Georgia Tech to Duke University in January 2018, has shown that TiO2 NPs, in the absence of light, produce reactive oxygen species (ROS) that oxidize proteins in contact with the TiO2 NPs. The oxidized proteins initiate an oxidative stress response in cells. These previous experiments were carried out using blood serum proteins and HeLa and A549 cells. The goal of this R21 is to translate these experiments to the lung, working in collaboration with Dr. Robert Tighe, a faculty member in Pulmonary, Allergy, and Critical Care Medicine at Duke?s School of Medicine and an expert in the pulmonary response to environmental pollutants. Experiments will progress from bronchoalveolar lavage fluid to airway macrophage and epithelial cells. Three types of TiO2 NPs, previously characterized in the Payne Lab, will be used for experiments; industrial-grade TiO2 NPs, surface passivated TiO2 NPs that do not produce ROS, and plasma- treated TiO2 NPs with increased ROS production. The significance of this research is obtaining a molecular- level, mechanistic, understanding of the interaction of TiO2 NPs with lung cells including a comparison of rodent and human samples. The innovative aspect of this research is a new in vitro method to explore cellular responses to NPs, including toxicity. This has implications beyond TiO2 NPs as this approach could be used to understand cellular responses to a broad array of industrial and environmental NPs. Since the majority of NP studies focus on cell culture conditions that do not represent actual in vivo conditions, this experimental design is a significant technical innovation. The outcome of this research will be a mechanistic understanding of TiO2 NPs in a pulmonary environment to define their true impact on human health. While risky, extensive Payne Lab studies of these NPs in model systems, coupled with Tighe Lab expertise in the pulmonary response to environmental exposures, makes the proposed work experimentally tractable. 0